Detection of analytes in biological samples is a common approach in the diagnostics and treatment area of medicine. In some cases, analytes requiring detection are bound to proteins in vivo, and samples submitted for detection of an analyte are difficult to process and accurately detect due to the interference of a protein bound to the analyte.
One such analyte is vitamin D. In humans, vitamin D exits in two forms—vitamin D2 and vitamin D3. Vitamin D2 (ergocalciferol) is obtained from dietary sources. Two forms of vitamin D can be obtained from dietary sources: vitamin D3 (cholecalciferol) is a 28-carbon molecule derived from the plant sterol ergosterol and humans obtain it from dietary sources. Vitamin D3 is a 27-carbon derivative of cholesterol and is synthesized in the epidermal layer of the skin via exposure to sunlight to cause photodegradation of 7-dehydrocholesterol.
Both forms of vitamin D, the endogenously-synthesized vitamin D3 and the diet-derived vitamin D2, are transported to the liver by proteins—principally the vitamin D binding protein and albumin. In the liver, the vitamin D forms are hydroxylated in the liver into 25OHD2 and 25OHD3, respectively (collectively, 25-hydroxyvitamin D (25OHD)). A further hydroxylation reaction of 25OHD occurs in the kidney to form the active hormone 1,25(OH)2D. Production of 1,25(OH)2D is tightly regulated by factors such as parathyroid hormone and phosphorus levels and it has a half-life of only about 4 to 6 hours. In comparison, 25OHD can be stored in muscles and adipose tissue and has a longer plasma half-life of about 2-3 weeks. The plasma concentration of 25OHD is measured as a primary indicator of in vivo vitamin D status.
Methods and assays for determining circulating vitamin D levels as indicated by 25OHD concentration have been developed, including high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), radioimmunoassay (RIA), chemiluminescent immunoassays (CLIA) and enzyme linked immunoassays (ELISA). There remains significant variability and an unfortunate lack of standardization in assessing vitamin D status (Binkley et al., J Clin Endocrinol Metab, 89:3152-3157 (2004)). One of the problems encountered in vitamin D measurement is the hydrophobic nature of 25OHD and to the fact that most of circulating 25OHD is bound to protein, with about 88% of circulating 25OHD bound to vitamin D binding protein (DBP) and about 12% bound to albumin (Hollis, B., Am J Clin Nutr, 88:507S-510S, (2008)). Methods for measuring circulating levels of vitamin D and for processing samples for measuring vitamin D levels in a human sample are needed.
Vitamin D is just one example of analytes in vivo that may be bound to a protein that interferes with the ability to detect and/or quantify the analyte. The methods described herein provide a solution for detection of an analyte separate from one or more binding proteins.
The foregoing examples of the related art and limitations related therewith are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification and a study of the drawings.